Signal transduction in vision involves a series of proteins that are sequentially activated via protein-protein interactions, thus passing the signal from the receptor to a final target. The long-term objectives of this project are to elucidate the molecular mechanisms of activation and signal propagation in three proteins of the relay: rhodopsin, transducin and arrestin. Of particular interest is the role of dynamics in function, i.e. the roles of backbone flexibility in protein-protein interactions and of conformational flexibility in activation. To obtain information on structure and dynamics in the complex membrane environment of signal transduction, the technique of site-directed spin labeling (SDSL) will be further developed. In this approach, nitroxide side chains are introduced at specific sites in a protein and analyzed by electron paramagnetic resonance (EPR) spectroscopy. Specific aims include: (1) analysis of nitroxide side chain interactions and dynamics via X-ray crystallography and spectral simulations, (2) evaluation of multifrequency EPR methods and new nitroxide side chains for analysis of backbone flexibility, and (3) evaluation of SDSL and high pressure EPR for detection of conformational flexibility in proteins. Modern pulse-EPR methods including saturation recovery, p-ELDOR, 2D-ELDOR and DEER will play important roles. These and existing SDSL tools will be applied to elucidate structure and dynamics in: (1) the conformational substates of activated rhodopsin (R*), (2) transducin in the R*-transducin complex and the mechanism of its activation, and (3) arrestin in solution and in the R*-arrestin complex, and the transformations leading to the high-affinity binding form. A growing number of pathological conditions have been traced to genetic defects in the proteins of the visual and other signaling systems. In order to design pharmacological approaches for altering such pathological states, it is imperative to understand molecular mechanism involved in the signal relay. Success in this project is anticipated to contribute to this understanding.